The immune responses to Pneumocystis are poorly understood, but cytokines may play a role in both clearing Pneumocystis infection and in the hypoxia associated with Pneumocystis pneumonia that may be exacerbated following initiation of therapy. We are using immunodeficient mouse models, especially the CD40L KO mouse, as well as immunocompetent C57BL/6J mice to further evaluate the role of individual cytokines and other immunoregulatory molecules in modulating Pneumocystis infection. We have developed a real time PCR assay to quantitate Pneumocystis over a wide dynamic range, and are currently examining Pneumocystis infection in healthy animals to better understand immune responses in the normal host. Using microarrays, we have been able to identify genes that are differentially expressed during Pneumocystis infection in healthy compared to immunodeficient mice; the majority of differentially expressed genes are related to the immune system. Immunohistochemical studies of the lungs done in parallel have demonstrated that there is an influx of CD4 cells and macrophages at 5 weeks, and, surprisingly, an influx of B cells at 6 weeks in immunocompetent mice but not CD40L KO mice. Microarray studies suggest that the B cells persist through at least 10 weeks, well after the infection has been cleared. We have extended these studies to examine immune responses in other immunodeficient mice, including those with defective innate immune responses, such as Myd88 knockout mice which are deficient in TLR signals, and CD1d KO mice, which lack NKT cells. Our studies have shown that these mice are not susceptible to uncontrolled Pneumocystis infection, suggesting that neither TLR signaling, nor NKT cells are critical to controlling Pneumocystis infection. In collaboration with Philip Murphy, Michail Lionakis, and their groups, we have examined changes in expression of a variety of chemokine and chemokine receptor genes, using Taqman real-time PCR assays, and have been able to identify CCR2 and its ligands as potentially important in the response to Pneumocystis infection. However, in examining the susceptibility of CCR2 knock-out mice to Pneumocystis infection, we found that they are able to clear infection similar to wild-type animals. We also examined CX3CR1 knock-out mice for susceptibility to Pneumocystis infection, since CX3CR1 and its ligand identify alternately activated macrophages and are critical to clearance of another fungus, Candida, in mouse models. These mice were again able to clear Pneumocystis infection with kinetics similar to wild-type mice. Using microarray analysis of purified CD4 cells isolated from lungs of Pneumocystis-infected mice, we were able to show that CXCR6 is preferentially upregulated compared to cells from uninfected animals, and by Q-PCR that both CXCR6 and its ligand, CXCL16, are unregulated in lung tissue from immunocompetent Pneumocystis-infected mice. We are currently examining the role of CXCR6 in controlling Pneumocystis infection through use of CXCR6 knock-out mice. We are examining the kinetics of immune responses in the lung of healthy animals exposed to Pneumocystis, using flow cytometry. We have found that interferon-gamma, and less predictably, IL-17, expression are increased in lymphocytes of the lung, primarily CD4 cells, during Pneumocystis infection and clearance, suggesting that Th1 and Th17 CD4+ T cells may play a role in clearing infection. Immunodeficient CD40L KO mice did not show changes in either of these populations during Pneumocystis infection. In follow-up, we found that IL-17A knockout mice are able to clear Pneumocystis infection, thus demonstrating that IL-17 is not critical to control of Pneumocystis infection. We have extended these studies to demonstrate that Th2 and Treg cells show minimal changes in in the lung during Pneumocystis infection in healthy mice, and that an anti-interferon-gamma antibody administered to immunocompetent mice causes a shift from Th1 to Th17 responses, but does not impact control of infection., with kinetics of clearance that are similar to untreated controls. We have recently examined the contribution of beta-glucans in cysts to the inflammatory response to Pneumocystis. We have demonstrated that, similar to other pathogenic fungi, glucans are to a large extent masked by surface proteins in 3 Pneumocystis species (P. murina, P. carinii, and P. jirovecii). Moreover, we have shown, by comparing infected mice treated with caspofungin, an inhibitor of beta 1,3 glucan synthase, to untreated mice, that beta-glucans are major contributors to the inflammatory response to Pneumocystis. We are also examining the response of dendritic cells to the most abundant Pneumocystis antigens, the major surface glycoprotein (MSG). We found that MSG did not activate dendritic cells, as assessed by surface markers, cytokine production, and microarray analysis. We hypothesize that this is because Pneumocystis cannot produce high mannans, which are important activators of innate immune responses through binding to C-type lectins, because they have lost the enzymes needed for such high mannosylation. Of note however, Msg was able to bind to 2 relevant C-type lectins, DC-SIGN and macrophage mannose receptor. We are following up on these initial studies to better characterize the interaction between dendritic cells and MSG, specifically to determine if MSG modulates the interaction of dendritic cells with beta glucans, which are present in the cyst wall. Finally, we are performing reconstitution studies in CD40KO mice, to try to identify which cells are critical to the mounting an effective immune response against Pneumocystis. For these studies we use CD45 allelic variants as donors to allow us to track transferred cells. We have been able to show that unfractionated spleen cells from immunocompetent mice are able to clear Pneumocystis infection, and are currently examining which subpopulations of cells can confer this protection. Preliminary studies suggest that B cells play an important role in conferring long-lasting protection. We are further examining the role of B cells by performing immune reconstitution studies using B cells that are quasi-monoclonal, to help determine if antibody production or, alternatively, other properties of B cells, such as antigen presentation, are critical to controlling Pneumocystis infection. It is hoped that these studies will provide insights into the mechanisms critical for control of Pneumocystis infection in the healthy host, and may provide mechanisms for increasing clearance of Pneumocystis or decreasing the inflammation that may be causing hypoxia.